Cytochrome P450 reductase is part of the ω-hydroxylase enzyme complex that catalyzes the first step in the ω-oxidation pathway. Cytochrome P450 reductase (CPR) catalyzes the reduction of the heme iron of various cytochromes, including cytochrome c and cytochrome P450. CPR has been renamed in recent literature as NADPH:cytochrome P450 oxidoreductase (NCP). The present application however, refers to the enzyme by its older designation, CPR. In the case of cytochrome P450s, this reduction initiates the catalytic steps that activate oxygen and result ultimately in the oxidation of substrates. In C. tropicalis, one class of cytochrome P450s catalyzes the ω-hydroxylation of fatty acids and the α,ω-hydroxylation of alkanes. Subsequent reactions by other enzymes generate α,ω-dicarboxylic acids, which are useful as raw materials for the preparation of numerous products such as perfumes, polymers, adhesives, coatings, lubricants, and macrolid antibiotics.
Cytochrome P450s are heme thiolate proteins consisting of a heme moiety bound to a single polypeptide chain of 45,000 to 55,000 daltons (Da). The iron of the heme prosthetic group is located at the center of a protoporphyrin ring. Four ligands of the heme iron can be attributed to the porphyrin ring. The fifth ligand is a thiolate anion from a cysteinyl residue of the polypeptide. The sixth ligand is probably a hydroxyl group from an amino acid residue, or a moiety with a similar field strength such as a water molecule as described, e.g., in Goeptar et al., Critical Reviews in Toxicology 25(1):25–65 (1995), incorporated herein by reference.
Monooxygenation reactions catalyzed by cytochrome P450 in a eukaryotic membrane-bound system require the transfer of electrons from NADPH to cytochrome P450 via NADPH-cytochrome P450 reductase (CPR) as described, e.g., in Taniguchi et al., Arch. Biochem. Biophys. 232:585 (1984), incorporated herein by reference. CPR is a flavoprotein of approximately 78,000 Da containing 1 mol of flavin adenine dinucleotide (FAD) and 1 mol of flavin mononucleotide (FMN) per mole of enzyme as described, e.g., in Potter et al., J. Biol. Chem. 258:6906 (1983), incorporated herein by reference. The FAD moiety of CPR is the site of electron entry into the enzyme, whereas FMN is the electron-donating site to cytochrome P450 as described, e.g., in Vermilion et al., J. Biol. Chem. 253:8812 (1978), incorporated herein by reference. The overall reaction is as follows:H++RH+NADPH+O2→ROH+NADP++H2O
Binding of a substrate to the catalytic site of cytochrome P450 apparently results in a conformational change initiating electron transfer from CPR to cytochrome P450. Subsequent to the transfer of the first electron, O2 binds to the Fe2+-P450 substrate complex to form Fe3+-P450-substrate complex. This complex is then reduced by a second electron from CPR, or, in some cases, NADH via cytochrome b5 and NADH-cytochrome b5 reductase as described, e.g., in Guengerich et al., Arch. Biochem. Biophys. 205:365 (1980), incorporated herein by reference. One atom of this reactive oxygen is introduced into the substrate, while the other is reduced to water. The oxygenated substrate then dissociates, regenerating the oxidized form of the cytochrome P450 as described, e.g., in Klassen, Amdur and Doull, Casarett and Doull's Toxicology, Macmillan, New York (1986), incorporated herein by reference.
The natural CPR proteins contain a hydrophobic N-terminal domain that anchors to membranes, resulting in localization of the protein in the microsomal fraction of cells, not in the cytoplasmic fraction that contains freely soluble proteins. A soluble CPR from rat has been produced recombinantly in yeast by Yabusaki et al. 1988, who reported a 33-fold enhancement of activity over the membrane-bound form. When adjusted for the observed 20-fold higher expression of the protein (based on antibody binding) however, the intrinsic enhancement of soluble CPR activity was less than 2-fold. A soluble CPR from S. cerevisiae has also been reported by Venkateswarlu et al. 1998. This soluble CPR had approximately the same activity as the microsomal form.
A full length CPR purified from C. maltosa in the presence of detergents, has a reported final specific activity of only about 63 U/mg (Scheller et al. 1996). Values reported for full-length CPRs purified from other sources are similar to those for C. maltosa, ranging from 50–70 U/mg protein (Dignam and Strobel, 1977; Yasukochi and Masters, 1976). Masters et al. have determined the specific activities of the purified full-length rat liver CPR and its proteolytically solubilized derivative to be almost identical, approximately 60 U/mg (Masters et al., 1975; Yasukochi and Masters, 1976).
The aforementioned activities are for reduction of cytochrome c, not for the oxidation of a substrate in P450-dependent reactions. In the rat and S. cerevisiae examples, the soluble CPRs were about 80% as active in reactions coupled to the hydroxylase activity of cytochrome P450s (Lamb et al., 1999; Venkateswarlu et al., 1998; Yabusaki et al., 1988).
Previous work in Candida tropicalis has demonstrated that strains genetically engineered to contain multiple copies of a full-length reductase gene, result in increased diacid production. See U.S. Pat. No. 6,331,420. Host cells engineered to produce one or more copies of a soluble form of reductase having activity in cytochrome P450-dependent reactions are highly desirable for several reasons. An enzymatically active, soluble CPR will not compete for space in the membranes, therefore allowing higher production of the membrane associated cytochrome P450s and fatty alcohol oxidase genes. In addition, less stress will be placed on the host cells. The present invention provides compositions and methods related to the production of a highly active, soluble form of cytochrome P450 reductase (CPR) from Candida tropicalis. 